Immunoassay cuvettes

ABSTRACT

The present invention is directed immunoassay cuvettes that comprise diffusely bound and non-diffusely bound reagents for carrying out an immunoassay. The reaction and detection are carried out in the immunoassay cuvette. The immunoassay cuvette comprises a transparent front wall, a back wall, side walls, a bottom, and a top opening. The back wall of the cuvette has a substantially planar surface made of a non-porous material and comprises a capture zone having reagents non-diffusedly bound and a signal reagent zone having reagents diffusedly bound.

This application claims the benefit of U.S. Provisional Application No.61/027,749, filed Feb. 11, 2008, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an immunoassay cuvette that comprisesdiffusely bound and non-diffusely bound reagents for carrying out animmunoassay. The reaction and detection are carried out in theimmunoassay cuvette.

BACKGROUND OF THE INVENTION

A bench top immunoassay analyzer is considered to have medium samplethroughput capability falling between POC (point of care) and highvolume analyzers. Where POC instruments measure 1 or 2 samples at a timeand high volume systems are capable of handling hundreds to thousands ofsamples, bench top analyzers with medium throughput can accommodatesomewhere around the range of ten to one hundred samples. The term“bench top” used to describe these medium throughput analyzers indicatesthe instruments are intended to be small enough to be placed in small tomid sized clinics on laboratory benches.

One challenge to the design of a bench top analyzer is that someclinical situations demand multi-analyte capability where severalmarkers are measured in the same sample. Multi-analyte capability canoffer the user more rapid result turn around time and greaterconvenience with lower cost compared to running individual assays foreach marker in the panel. A bench top analyzer should be capable ofoffering a full menu of immunoassays (over 50 assays) and many of theseassays have different protocols, assay times and reagents, all of whichdemand that the instrument system including the consumables (disposablereagents and immunoassay devices) have the flexibility to accommodatethese differing assay requirements. Another challenge is that inclinical labs, bench space is at a premium and the “foot print” of aninstrument is a major consideration.

Clinical immunoassay analyzers offer a full menu of assays; they need tohave all or many of the assay reagents in the menu residing in theinstrument in order to be available when test panels are ordered. Thestorage of multiple reagent vials adds to the space and complexity ofthe instrument system. The fluid handling subsystem must be capable ofwithdrawing an aliquot from the reagent vial and dispense it into a testdevice along with sample. Often to achieve long term stability of theassay reagents, the storage chamber is be refrigerated. FIG. 1 shows atypical bench top analyzer manufactured by Adaltis with the reagentvials, cuvette tray and the major subsystems within the instrument. Amajor portion of the instrument illustrated in FIG. 1 is dedicated tostorage of reagents and calibrators necessary to offer a full menuassays.

Since consumables (reagents and immunoassay devices) are central to thedesign of an immunoassay instrument system, there is a need for unitizedconsumables that keep all the essential reagents in one unit and in adry format. The unitized consumables improve the reagent stability,reduce the complexity and overall size of a bench top analyzer, and addto user convenience.

SUMMARY OF INVENTION

The present invention is directed to immunoassay cuvettes comprising atransparent front wall, a back wall, side walls, a bottom, and a topopening.

In one embodiment, the immunoassay cuvette is suitable for a one-stepimmunoassay either in a competitive format or a sandwich format. In thisembodiment, the back wall of the cuvette has a substantially planarsurface made of a non-porous material and comprises a capture zone and asignal reagent zone. The capture zone comprises one or more spots eachnon-diffusely bound with a first member of a binding pair. The signalreagent zone having one or more spots diffusely bound with a signalreagent comprising the first member or a second member of the bindingpair.

In another embodiment, the immunoassay cuvette is suitable for atwo-step sequential binding immunoassay either in a competitive formator a sandwich format, when the analyte is an antigen. In thisembodiment, the back wall of the cuvette has a substantially planarsurface made of a non-porous material and comprises a capture zone, abinding reagent zone, and a signal reagent zone. The capture zonecomprises one or more spots non-diffusely bound with a first antibodyagainst a defined antigen. The binding reagent zone comprises one ormore spots diffusely bound with a binding reagent comprising (a) thedefined antigen or (b) a second antibody against the defined antigen.The signal reagent zone has one or more spots each diffusely bound witha signal reagent comprising a conjugate of a signal generating moleculeand a binding molecule that binds to the binding reagent but does notbind to the first antibody. The signal reagent zone is located above thebinding reagent zone and the capture zone.

In another embodiment, the immunoassay cuvette is suitable for atwo-step sequential binding immunoassay either in a competitive formator a sandwich format, when the analyte is an antibody. In thisembodiment, the back wall has a substantially planar surface made of anon-porous material comprising a capture zone, and a signal reagentzone. The capture zone comprises one or more spots each non-diffuselybound with a defined antigen. The signal reagent zone having one or morespots diffusely bound with a signal reagent comprising an antibodyagainst a human immunoglobulin such as IgG or IgM, wherein the signalreagent zone is located above the capture zone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a prior art bench top analyzer.

FIG. 2 shows a one-step binding reaction in a sandwich assay, where theanalyte is an antigen.

FIG. 3 shows a one-step binding reaction in a sandwich assay, where theanalytes are multiple antigens.

FIG. 4 shows a one-step binding reaction in a competition assay, wherethe analytes are multiple antigens.

FIG. 5 shows a two-step sequential binding reaction in a sandwich assay,where the analyte is an antigen.

FIG. 6 shows a three-step sequential binding reaction in a sandwichassay, where the analyte is an antigen.

FIG. 7 shows vertical positioning of immunoassay cuvettes.

FIG. 8 shows reagent aspiration in an immunoassay cuvette.

FIG. 9 shows the design of fluorescence detection in an immunoassaycuvette.

FIG. 10 shows an multiple analyte format in two chambers.

FIG. 11 shows the two-part assembly of an immunoassay cuvette.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A binding pair, as used herein, refers to two molecules that areattracted to each other and specifically bind to each other. Examples ofbinding pairs include, but not limited to, an antigen and an antibodyagainst the antigen, a ligand and its receptor, complementary strands ofnucleic acids, biotin and avidin, biotin and streptavidin.

Diffusively bound, as used herein, refers to reagents deposited in a dryformat on a solid phase. When a liquid sample is added to the solidphase, such reagents become free to diffuse in the liquid medium.Examples of diffusively bound reagents in this application includereagents in the signal reagent zone and binding reagent zone.

Immobilized, as used herein, refers to reagents being fixed to a solidsurface. When a reagent is immobilized to a solid surface, it is eitherdiffusely bound or non-diffusely bound to the surface.

Non-diffusively bound, as used herein, refers to reagents immobilized toa solid phase and are not free to diffuse in a liquid medium. Examplesof non-diffusively bound reagents in this application are reagents inthe capture zone.

A signal generating molecule refers to a molecule that can generatesignals for detection. For example, a signal generating molecule is afluorescent dye, a chemiluminescent dye, or an enzyme (e.g. alkalinephosphatase or β-galactosidase) that can react on a substrate togenerate signals.

A non-porous material, as used herein, refers to a material that hasless than 20%, preferably less than 10%, preferably less than 5%, mostlypreferably less than 1% of its volume occupied with void spaces. Anon-porous material includes, but not limited to plastics such aspolymethylmethacrylate, polystyrene, polycarbonate, glass, quartz, orcombination thereof.

The present invention provides various formats of immunoassay cuvettessuitable for a bench top clinical immunoassay analyzer. The immunoassaycuvettes are unitized consumables that contain all the essentialreagents for immunoassays in a dry format. Unlike prior art bench topanalyzer, which reagents and calibrators have to be stored refrigeratedwithin the analyzer, the immunoassay cuvettes have excellent long termstability and they do not have to be stored within the instrument atrefrigerated temperature. The immunoassay cuvettes keep incompatiblereagents separated, for example, in capture zone and reagent zone untilthe assay is started.

The immunoassay cuvette of the present invention is a reaction chamberdesigned for one or more immunoassay reactions, which provide a signalfor detecting analytes. The immunoassay cuvette comprises a transparentfront wall, a back wall, side walls, a bottom, and a top opening. Theback wall has a substantially planar surface made of a non-porousmaterial, which the essential reagents are immobilized on.

One-Step Binding Reaction

This format has a capture zone (non-diffusely bound) and reagent zone(diffusively bound), which work for one binding step assays, i.e. noneed for sequential binding. The prime advantage of sequential bindingis that it can amplify signals and improve sensitivity; however, notevery assay requires high sensitivity or needs amplification. For theone step assays, the diffusively bound reagent is labeled with a signalgenerating molecule such as a fluorescent dye. Another feature of thisformat is that it keeps incompatible reagents separated in capture andreagents zones until the assay is started. This is mandatory forcompetitive assays.

The immunoassay cuvette for a one-step binding reaction can be designedin a competitive assay format or in a sandwich assay format. The backwall of the immunoassay cuvette comprises a capture zone and a signalreagent zone. The capture zone comprises one or more spots eachnon-diffusely bound with a first member of a binding pair, and thesignal reagent zone having one or more spots diffusely bound with asignal reagent comprising the first member or a second member of thebinding pair. In this embodiment, when the signal reagent comprises thefirst member of the binding pair, it is a sandwich assay. When thesignal reagent comprises the second member of the binding pair, it is acompetitive assay.

In one embodiment, the immunoassay cuvette has a capture zone thatcomprises 1 to 20 spots, preferably 1-8 spots, each non-diffusely boundwith a first member of a binding pair, such as an antibody. In amultianalyte format, spots can be dedicated, for example, to members ofa binding pair, to serve as a procedural control. A procedure control inthis case can be an antigen/antibody pair that does not occur in humansamples. A binding reaction can then be constructed to give a consistentfluorescent signal from sample to sample. The procedural control thenacts to monitor whether the instrument performed the assay correctly, ifreagents remained stable during storage, or if the correct cuvette wasused. Examples of antigen/antibody pairs suitable of procedural controlswould be fluorescein/anti-fluorescein anddinitrophenol/anti-dinitrophenol. One member of the procedural controlbinding pair is non-diffusively bound in a spot in the capture zone andthe second member, labeled with a fluorescent tag, can be diffusivelybound in either the binding reagent zone or signal reagent zone. In apreferred embodiment, the signal reagent zone has only one spot.

Sandwich Assay Format

FIG. 2 illustrates the immunoassay cuvette designed for a sandwich assaywhere the analyte is an antigen. In the capture zone, the spot isnon-diffusely bound with a first antibody against the antigen. In thesignal reagent zone, the spot is diffusely bound with a signal reagentcomprising a second antibody against the antigen. The first and thesecond antibody can be identical or different. When the first and thesecond antibody are different, they can be monoclonal antibodies againstdifferent epitopes, or they can be different polyclonal antibodies, orthey can be monoclonal antibody and polyclonal antibody. In FIG. 2, theantibody is label with a signal generating molecule such as afluorescent dye (S-Ab). When the antigen sample is added to the cuvette,the fluid height should be above both the capture zone and the signalreagent zone. The S-Ab is free to diffuse and binds to antigen captureby the antibody in the capture zone and provides a signal.

FIG. 3 and Example 2 illustrate the immunoassay cuvette designed forsandwich assays for multiple antigen analytes. In the capture zone, spot1 is non-diffusely bound with a first antibody against Antigen 1,myoglobin. Spot 2 is non-diffusely bound with a first antibody againstAntigen 2, troponin I. In the signal reagent zone, spot 1 is diffuselybound with a signal reagent comprising a second antibody against Antigen1 and a second antibody against Antigen 2. The antibodies are labeledwith a signal generating molecule such as a fluorescent dye (S-Ab). Whenthe antigen sample is added to the cuvette, the fluid height is designedto be above both the capture zone and the signal reagent zone. Thesignal labeled-antibodies are free to diffuse and bind to Antigen 1 andAntigen 2 captured by its respective antibody in the capture zone andprovide a signal in spot 2 and spot 3.

Competitive Assay Format

In a one step binding, competitive assay format for an antigen analyte,the capture zone has the antigen non-diffusedly immobilized. The signalreagent zone is diffused bound with the antibody labeled with a signalgenerating molecule. When the antigen sample is added to the cuvette,the fluid height should be above both the capture zone and the signalreagent zone. The signal labeled-antibody is free to diffuse and bindseither to the antigen in the sample or the antigen in the capture zone.

In a one step binding, competitive assay format for an antigen analyte,the capture zone has the antibody non-diffusedly immobilized. The signalreagent zone is diffusedly bound with the antigen labeled with a signalgenerating molecule. When the antigen sample is added to the cuvette,the fluid height should be above both the capture zone and the signalreagent zone. The signal labeled-antigen is free to diffuse and competewith the antigen in sample for binding to the antibody in the capturezone.

FIG. 4 and Example 3 illustrate the immunoassay cuvette designed forcompetitive assays for multiple antigen analytes. In the capture zone,spot 3 is non-diffusely bound with Antigen 1, e.g., myoglobin. Spot 2 isnon-diffusely bound Antigen 2, e.g., troponin I. In the signal reagentzone, spot 1 is diffusely bound with a signal reagent comprising anantibody against Antigen 1 and an antibody against Antigen 2. Theantibodies are labeled with a signal generating molecule such as afluorescent dye (S-Ab). When the antigen sample is added to the cuvette,the fluid height should be above both the capture zone and the signalreagent zone. The signal labeled-antibodies are free to diffuse and bindeither to the Antigens 1 and 2 in the sample or the Antigens 1 (spot 3)and 2 (Spot 2) in the capture zone.

Two-Step Sequential Binding Reaction

Sequential format are suitable for assays that need high sensitivity.The sequential format offers a two-step sequential binding protocol,where the second step has a signal amplification reagent that binds tocomplexes in the capture zone. The reason why a two step bindingprotocol is needed is that if a signal reagent is present in the firstbinding step, it will interfere with the formation of the immune complexin the capture zone. The immune complex must be formed first before thesignal reagent binds; therefore a sequential protocol is required. Thesequential format is suitable for both sandwich and competitive bindingassays in single and multi-analyte modes.

Antigen Analyte

In this embodiment, the immunoassay cuvette is designed for a two-stepbinding reaction either in a competitive assay format or in a sandwichassay format. The back wall of the immunoassay cuvette comprises acapture zone, a binding reagent zone, and a signal reagent zone.

The capture zone comprises one or more spots non-diffusely bound with afirst antibody against a defined antigen. The binding reagent zonecomprises one or more spots diffusely bound with a binding reagentcomprising (a) the defined antigen (for a competitive assay format) or(b) a second antibody against the defined antigen (for a sandwich assayformat). (a) or (b) can be labeled with a first member of a binding pairsuch as biotin/streptavidin.

The signal reagent zone has one or more spots diffusely bound with asignal reagent comprising a conjugate of a signal generating moleculeand a binding molecule that binds to the binding reagent but does notbind to the first antibody. The binding molecule either binds to thesecond antibody or binds to the first member of the binding pair. Thesignal reagent zone is located above the binding reagent zone and thecapture zone.

FIG. 5 illustrates the immunoassay cuvette designed for a two-step,sequential binding sandwich assay, where the analyte is an antigen. Thecapture zone comprises one or more spots non-diffusely bound with afirst antibody against a defined antigen. The binding reagent zonecomprises one or more spots diffusely bound with a binding reagentcomprising a second antibody against the defined antigen. The signalreagent zone has one or more spots diffusely bound with a signal reagentcomprising a conjugate of a signal generating molecule and a bindingmolecule that binds to the binding reagent (e.g., the second antibody)but does not bind to the first antibody. The signal reagent zone islocated above the binding reagent zone and the capture zone. When anantigen sample is added, the fluid height is below the signal reagentzone, and the second antibody is free to diffuse and bind to the antigencaptured in the capture zone. After washing to remove the unboundmaterials, a buffer is added to above the height of the signal reagentzone. The signal reagent is dissolved in the buffer and freely todiffuse and bind to complex in the capture zone.

FIG. 5 illustrates the fluid management with the Unitized Consumable toconduct assays with sequential reactions. Diffusively bound andnon-diffusively bound reagents are brought into reaction when thererespective zones are hydrated by addition of a diluent reagent to thecuvette. Since the zones are positioned at specified heights on thevertical wall, sequential reactions can be performed by increasing thevolume of diluent reagent thereby hydrating zones at higher locations onthe cuvette wall. FIG. 5 is an example of a sandwich immunoassay withone antibody non-diffusively bound in the capture zone and the secondantibody diffusively bound in the reagent binding zone. Addition of adiluent reagent containing sample antigen initiates the first bindingreaction. The volume of the diluent reagent is such that the fluidheight is below the Signal Reagent Zone. Since most sandwichimmunoassays are heterogeneous, the immunoassay cuvette is flexible forincubation times and number of wash cycle as long as the fluid height ismaintained below the Signal Reagent Zone. At the completion of the firstbinding reaction, the second reaction is started by adding a volume ofdiluent reagent so that the fluid height is higher than the signalreagent zone, which in this case hydrates a fluorescent binding reagentthat can bind to immune complexes in the capture zone. After incubationperiod and wash cycle, fluorescence in the capture zone is measured.

Three-Step Sequential Binding Reaction, Antigen Analyte

In a three-step assay, the immunoassay cuvette's design is similar tothat of the above-described for a two-step assay, except the bindingreagent zone is located above the capture zone.

FIG. 6 illustrates the immunoassay cuvette designed for a three-step,sequential binding sandwich assay, where the analyte is an antigen. Thecapture zone comprises one or more spots non-diffusely bound with afirst antibody against a defined antigen. The binding reagent zonecomprises one or more spots diffusely bound with a binding reagentcomprising a second antibody against the defined antigen. The signalreagent zone has one or more spots diffusely bound with a signal reagentcomprising a conjugate of a signal generating molecule and a bindingmolecule that binds to the binding reagent (e.g., the second antibody)but does not bind to the first antibody. The signal reagent zone islocated above the binding reagent zone, and the binding reagent zone islocated above the capture zone. When an antigen sample is added, thefluid height is below the binding reagent zone, and the antigen binds tothe antibody in capture zone. After washing, fluid is added to below thesignal reagent zone to hydrate the binding reagent, which is freelydiffused and binds to the complex in the capture zone. After washing,fluid is added to hydrate the signal reagent, which is freely diffusedand binds to the complex in the capture zone.

Two-Step Sequential Binding Reaction, Antibody Analyte, Sandwich Format

Another format of an immunoassay cuvette is designed for a sandwichassay where the analyte is an antibody, e.g., a serological assay for anantibody against hepatitis surface antigen such as hepatitis B orhepatitis C antigen. In this format, the capture zone comprises one ormore spots each non-diffusely bound with a defined antigen. The signalreagent zone comprises one or more spots each diffusely bound with asignal reagent comprising an antibody against a human immunoglobulin(IgG or IgM), such as mouse anti-human IgG, wherein the signal reagentzone is located above the capture zone. In the signal reagent zone, theanti-immunoglobulin antibody is labeled with a signal generatingmolecule (such as fluorescent dye, enzyme, etc.) and there is no furtheramplification of the signal.

Alternatively, if the amplification of the signal is desired, a furthersignal reagent zone 2 above the first signal zone is provided. Theanti-immunoglobulin antibody in the first signal reagent zone is labeledwith a first member of a binding pair such as biotin/streptavidin, andthe second signal zone comprises a conjugate of a signal generatingmolecule and a second member of the binding pair.

Manufacturing

Development of diffusively bound reagents on vertical walls of cuvettereaction chambers with smooth, non-porous surfaces requires thecombination of several features. The reagents must be in a dry formatand adhere firmly enough to the surface during manufacture, shipment andmanipulation by users, until at the point of application. At a definedpoint in the assay protocol when the zone containing the diffusivelybound reagent is immersed in a diluent, the reagent must rapidlysolubilize, be free to diffuse in the liquid phase, and provide itsintended biological or chemical activity. Stabilization of proteinreagents, particularly antibodies, in a dry format, often requiresadditives to the protein formulation. Monomeric sugars, polysaccharides,and polyols are typical stabilizing additives. Selection of specificstabilizers in general is dependent on the particular assay format anddrying method utilized. For example, a reagent containing sucrose atconcentration ranging around 5-15% is sufficient to provide long termstability of the protein activity in a dry state, to adhere to avertical wall of cuvettes and to solubilize rapidly in about 1 minute.

Although diffusively bound reagents are commonly applied in lateral flowtest devices, such reagents are not suitable for application tonon-porous vertical walls. Lateral flow devices introduce fluid bycapillary action; reagent formulations are optimized to rapidlysolubilize, in a matter of seconds, without perturbing the capillaryflow. Adherence to the matrix is not an issue since lateral flow devicesare constructed with membranes or fibrous materials that provide ampleporosity and surface area to entrap the dried reagent. Conversely,formulations that are suitable for the vertical wall of the UnitizedConsumable are not suitable for lateral flow device since they will notsolubilize rapidly enough and will disrupt the uniformity of thecapillary flow.

Methods to immobilize reagents to the solid phase are common inimmunochemistry and involve formation of covalent, hydrophobic orelectrostatic bonds between the solid phase and reagent. Non-diffusivelybound reagents can be absorbed or covalently bound to the solid surface.Although the same adsorption method is used for immobilizing diffusivelybound reagents, a large excess of carrier protein is present on thespotting reagent formulation to competitively inhibit adsorption of thespecific reagent to the surface. Immobilization of non-diffusely boundand diffusely bound reagents on the back wall of an immunoassay cuvetteis illustrated in Examples 2-5 in details.

Advantages of Immunoassay Cuvettes

The immunoassay cuvette is formatted to contain all of the reagentsnecessary to perform immunoassays for one or multiple biomarkers in asingle sample using a common reaction chamber. Only addition of sampleand a common diluent reagent are necessary to perform an assay. Becausethe assay reagents are contained within a cuvette, the reagent storageand dispensing are eliminated from the bench-top analyzer (see FIG. 1),thus reducing the overall size of the analyzer.

Protein arrays are often glass slide based and positioned on ahorizontal plane within an instrument, which demands that a more complexclosed fluid system be implemented with connectors, tubing, valves etc.The immunoassay cuvette of the present invention has a single opening atthe top of the device; the opening is large enough for the insertionneedles for dispensing or aspiration of reagents. In general, only twofluid management stations are required with the immunoassay cuvette,i.e., a sample/diluent buffer dispense station and an aspiration washstation. The sample/buffer dispense station can deliver fluids ofspecific volume to rehydrate reagent zones of a specific height.

FIG. 7 shows a diagram of the immunoassay cuvette placed on a carousel,which illustrates another advantage of positioning the antibody capturezones in a vertical plane. In an analytical system designed to processmultiple samples, there must be a subsystem to hold samples andconsumables. This sample/consumable holding subsystem contributes to thesample capacity and foot print of the instrument system. Thesesample/consumable subsystems are often part of the mechanism to transferthe sample/consumables to different stations within the instrument toperform the assays. Carousels are used for radial motion and linked railmechanisms are used for linear motion. For bench top systems, there is acompromise between the maximum sample capacity and the minimum footprint.

The immunoassay cuvette preferably has a unique aspect ratio between itsheight, width and thickness dimensions, such that positioning thereagent zones on the vertical walls can be a more efficient use ofspace, as depicted in FIG. 7. This feature of the immunoassay cuvettemaximizes the sample capacity while minimizing the instrument footprint. Aspect ratio is defined as the ratio of height to the smallestdimension of either width or thickness. Aspect ratios around or greaterthan 3 to 1 are necessary to take advantage of space saving bypositioning reagent zones on the walls of the cuvette. As used herein,height of a cuvette refers to the distance between the bottom of thecuvette and the top opening. Thickness of a cuvette refers to thedistance between the front wall and the back wall. Width of a cuvetterefers to the distance between the sidewalls.

The aspect ratio of the immunoassay cuvette facilitates the fluidremoval (e.g., by aspiration) during the wash cycles of the assay. Washcycles employing aspiration can be problematic for solid phaseimmunoassays with small capture zones, particularly arrays. The problemrelates to the fact that the aspiration needle has to be brought intoproximity of the capture zone in order to effect efficient fluidremoval. At the end of the fluid removal, air flows into the needle athigh rates since the aspiration is based on negative pressure. The airflow can cause the capture zone to dry out and inactive any bindingreagent in the capture zone. Antibodies are especially prone todenaturation upon this type of drying. In situations where theaspiration needle is around 1 mm in diameter and the capture zone is acomparable dimension or smaller, a significant proportion of the bindingreagent can be inactivated which results in poor reproducibility andother performance problems with the assay. Although ELISA assays withmicroplates are commonly carried out with plate washers usingaspiration, some inactivation undoubtedly occurs, however, the effect isnot noticeable due to the relatively large surface area of the microwell(6 mm diameter at bottom and about 5 mm high side walls).

FIG. 8 shows the insertion of the aspiration needle to the bottom of theimmunoassay cuvette. With the capture zones on the vertical walls,removal of the fluid is achieved without locating the needle near thecapture zones, consequently avoiding denaturation of binding regents byair flow. The bottom of the immunoassay cuvette is preferred to besloped or curved. The sloping bottom with a cusp in the center/bottom ofthe device further enhances efficient fluid drainage and aspiration.

FIG. 9 diagrams one of the possible detection elements that can be usedin conjunction with the immunoassay cuvette. Detection employingfluorescent dyes is one preferred detection method although there areother possible techniques such as chemiluminescence and enzyme activity.Assays are designed to incorporate the fluorescent dye into the bindingcomplex formed in the capture zone when analyte is present. The backwall of the immunoassay cuvette that contains the antibody capture zonesis comprised of a plastic material that is formulated with a tintingagent that reduces the background fluorescence of the plastic material.Although alternative low fluorescence substrates (glass and quartz) canbe used, plastic is the most desirable since it can be molded at lowcost with the appropriate dimensions, surface features, etc. Standardplastics as PMMA and polystyrene have relatively high levels ofintrinsic fluorescence, which would be detected as background signal,thus they are limited in their application to high sensitivityfluorescence based solid phase immunoassays. The use of tinting agentscan reduce their fluorescence. FIG. 9 shows the top view of theimmunoassay cuvette and its orientation to the fluorescence detectionsubsystem comprising a laser diode for excitation and a photodetector tomonitor fluorescence emission. The front wall opposing the antibodycapture zones is made of a clear plastic material that allows for thefluorescence detection in the capture zones. The optics the detectionsystem are designed to minimize any possible contribution of the clearplastic to the fluorescence measurement.

Two Chamber Immunoassay Cuvettes

In an alternative embodiment of the invention, the immunoassay cuvettescomprise two chambers: a sample reaction chamber and a signal reagentchamber, separated by a partition wall. The sample chamber comprises afirst front wall, a first back wall, a first side wall, the partitionwall, a first bottom, and a first top opening. The first back wall hassubstantially planar surface, which comprises a capture zone, where thecapture reagents are non-diffusely bound, and optionally a bindingreagent zone with the binding reagent diffusively bound to thenon-porous wall of the sample chamber. The signal reagent chambercomprises a signal reagent zone, and optionally a binding reagent zone,where the signal reagents and binding reagents are diffusely bound tothe non-porous surface of the wall in the signal reagent chamber.

FIG. 10 depicts an assay format for multiple analyte detection in thesame sample using the two chamber immunoassay cuvette. The samplecontains two analytes where their respective antibodies are immobilizedin separate capture zones on the back wall of the sample reactionchamber. The second antibodies of the sandwich pairs are deposited inthe binding reagent zone. Both second antibodies are labeled with ahapten “H”. The purpose of the hapten H is to link immune complexesformed in the captures zones with an anti-H reagent labeled with afluorescent dye. The fluorescent anti-H is positioned in an adjacentsignal reagent chamber of the device. Sample is added to the samplereaction chamber to start the assay. After a brief incubation period,the sample chamber is washed several times to remove unbound antibody.Diluent is added to the signal reagent chamber and aAn aliquot of thefluorescent label anti-H is transferred to the sample reaction chamberand allowed to incubate followed by a wash sequence. Any immune complexformed between the antigen, immobilized antibody in the capture zone andthe antibody tagged with H will also have the fluorescent anti-H boundin that capture zone. The fluorescence detection subsystem can then scanthe capture zones to measure fluorescence.

EXAMPLES Materials

Human myoglobin, cat #1-023, Human troponin I, cat #1-020, andmonoclonal antibodies for human roponin I, clones A34440 & G-129, andhuman myoglobin, clones 7005 & 7004, were obtained from Biospacific(Emeryville, Calif.). Polyclonal antibody to Human Ig (G & M), cat#109-005-044, and monoclonal antibody to fluorescein (F), cat#200-002-0370, were obtained from Jackson. Immuno Research (West Grove,Pa.). The arylsulfonate cyanine dye, Cy5-NHS, cat #PA15101, was obtainedfrom GE Healthcare, (Piscataway, N.J.). Hepatitis B core antigen(HBcAg), cat #YVS8914, and Hepatitis B surface antigen (HBsAg), cat #OBT0912, were obtained foim Accurate Chemical and Scientific Corp(Westbury, N.Y.). Fluorescein isothiocyanate (FITC), cat #F143, was fromInvitrogen Corp. (Carlsbad, Calif.). Sucrose, BSA, phosphate bufferedsaline (PBS, pH 7.4), sodium carbonate, Tween 20, and sodium cholatewere obtained from Sigma-Aldrich Corp. (St. Louis, Mo.). Fluorescencedetection methodology is described in “Fluorescence Spectroscopy inBiology: Advanced Methods and their Application to Membranes, Proteins,DNA and Cells,” vol. 3, ed. Wolfbeis, pub. Springer, 2005.

Example 1 Label Antibodies with Cy5

Anti-fluorescein, anti-troponin I (clone G-129), anti-myoglobin (clone7004) and anti Ig(G&M) are labeled with Cy5-NHS as follows: To 1 mg. ofantibody in 1 ml of 0.1M sodium carbonate buffer, pH 9.0, is added 40 ugof Cy5-NHS. The mixture is allowed to reacted for 3 hours at roomtemperature. The unconjugated Cy 5 is removed from the antibody bychromatography on a Sephadex G25 column. Spectral analysis indicatestypically 2-4 Cy 5 molecules per antibody.

Anti-troponin I (clone G-129) and anti-myoglobin (clone 7004) arelabeled with FITC as follows: To 1 mg of antibody in 1 ml of 0.1M sodiumcarbonate, pH 9.0, is added 30 ug of FITC. The mixture is allowed toreact for 3 hours at room temperature, followed by chromatography on aSephadex G25 column to remove unconjugated FITC. Spectral analysisindicates around 2 FITC molecules per antibody.

Example 2 One-Step Sandwich Assay, Antigen Samples

With the back wall in the horizontal position, 3 μl of anti-troponin I(clone A34440) at 3 μg/ml in PBS is dispended in spot 2 and 5 μlanti-myoglobin (clone 7005) at 5 μg/ml in PBS is dispensed in spot 3.Spots 2 & 3 are designated as the capture zone (non-diffusively bound)The back wall is then placed in a humidified chamber and the proteinallowed to adsorb to the plastic for overnight at room temperature. Theprotein solution is then removed from spots 2 & 3 with a micropipette. Awash cycle is then performed by adding 3 μl of assay buffer comprised ofPBS, 1 mg/ml BSA and 0.1% Tween 20 to each of the spots. After about 10seconds the buffer is removed. The wash cycle is repeated 5 times. Afterthe last wash cycle 3 μl of a PBS buffer containing 10% sucrose and 1mg/ml BSA is placed in each of the spots. Following a 30 secondincubation, the sucrose/PBS buffer is removed from the spots leaving athin fluid film on the surface of the spots. A 3 μl aliquot containingCy5-anti-troponin I (clone G-129) and Cy5-anti-myoglobin (clone 7004),both at a concentration in 1 μg/ml in PBS buffer with 10% sucrose, 1mg/ml BSA, and 1% sodium cholate detergent is dispensed in spot 1. Spot1 is designated the signal zone (diffusively bound). The back wall,still in the horizontal position, is then placed in a 37 C convectionoven and allowed to dry of 1 hour. Epoxy is then placed along the rim tothe back wall and the front wall is bounded to the back wall to assemblethe unitized cuvette. The cuvette is stored desiccated until use.

To perform the sandwich assay, 5 μl of a sample containing humanmyoglobin and troponin is dispensed at the bottom of the cuvette with amicropipette. 45 μl of assay buffer is added, sufficient to immerse bothcapture and signal reagent zones (see FIG. 3). To facilitate mixing anddiffusion of the signal reagent, 20 μl of the mixture is withdrawn anddispensed rapidly back into the consumable with a micropipette,repeating several times over about a 10 second period. The assay mixturethen incubates for 20 minutes at room temperature, at which point theassay mixture is removed followed by 4 wash cycles with 70 μl of assaybuffer at each cycle. After the last wash cycle, 70 μl of assay bufferis added to the cuvette and the Cy 5 fluorescence is detected in spots 2& 3 to respectively measure troponin I and myoglobin in the sample.

Example 3 One-Step Competition Assay

FIG. 11 illustrates front and back walls of the unitized immunoassaycuvette prior to assembly. Both front and back walls are constructedwith polymethmethyl acrylate plastic. The immunoassay cuvette has aheight about 20-30 mm, a thickness about 1-10 mm (preferably 2-4 mm),and a width about 5-25 mm (preferably 5-15 mm). For example, theimmunoassay cuvette is 25 mm high, 12 mm wide, and 5 mm thick with aliquid capacity of about 140 μl. For reagent formatting, the back wallis horizontally positioned. Using a micropipette, 3 μl of troponin I(0.5 ug/ml PBS) is placed in spot 2 and 3 μl of myoglobin (0.5 ug/ml inPBS) is placed in spot 3. Spots 2 & 3 are designated as the capture zone(non-diffusively bound). The back wall is then placed in a humidifiedchamber and the protein allowed to adsorb to the plastic for overnite atroom temperature. The protein solution is then removed from spots 2 & 3with a micropipette. A wash cycle is then performed by adding 3 μl ofassay buffer comprised of PBS, 1 mg/ml BSA and 0.1% Tween 20 to each ofthe spots. After about 10 seconds the buffer is removed. The wash cycleis repeated 5 times. After the last wash cycle 5 μl of a PBS buffercontaining 10% sucrose and 1 mg/ml BSA is placed in each of the spots.Following a 30 second incubation, the sucrose/PBS buffer is removed fromthe spots leaving a thin fluid film on the surface of the spots. 5 μl ofa solution containing Cy5 anti-troponin I and Cy5-anti myoglobin, bothat a concentration in 1 μg/ml in PBS buffer with 10% sucrose and 1%sodium cholate detergent. Spot 1 is designated the signal zone(diffusively bound). The back wall, still in the horizontal position, isplaced in a 37 C convection oven and allowed to dry of 1 hour. Epoxy isthen placed along the rim to the back wall and the front wall is boundedto the back wall to assemble the unitized cuvette. The cuvette is storeddesiccated until use.

To perform the competitive assay, 5 μl of a sample containing humanmyoglobin and troponin is dispensed at the bottom of the cuvette with amicropipette. 45 μl of assay buffer is added, sufficient to immerse bothcapture and signal reagents zones, FIG. 4. To facilitate mixing anddiffusion of the signal reagent, 20 μl of the mixture is withdrawn anddispensed rapidly back into the consumable with a micropipette,repeating several times over about a 10 second period. The assay mixturethen incubates for 20 minutes at room temperature, at which point theassay mixture is removed followed by 4 wash cycles with 70 μl of assaybuffer at each cycle. After the last wash cycle, 70 μl of assay bufferis added to the consumable and the Cy 5 fluorescence is detected inspots 2 & 3 to respectively measure troponin I and myoglobin in thesample.

Example 4 Two-Step Sequential Protocol with Amplification (AntigenSamples)

With the back wall in the horizontal position, 3 μl of anti-troponin I(clone A34440) at 5 ug/ml in PBS is dispended in spot 2 and 3 μlanti-myoglobin (clone 7005) at 5 ug/ml in PBS is dispensed in spot 3.Spots 2 & 3 are designated as the capture zone (non-diffusively bound)The back wall is then placed in a humidified chamber and the proteinallowed to adsorb to the plastic for overnite at room temperature. Theprotein solution is then removed from spots 2 & 3 with a micropipette. Awash cycle is then performed by adding 3 μl of assay buffer comprised ofPBS, 1 mg/ml BSA and 0.1% Tween 20 to each of the spots. After about 10seconds the buffer is removed. The wash cycle is repeated 4 times. Afterthe last wash cycle 3 μl of a PBS buffer containing 10% sucrose and 1mg/ml BSA is placed in each of the spots. Following a 30 secondincubation, the sucrose/PBS buffer is removed from the spots leaving athin fluid film on the surface of the spots. A 3 μl aliquot containingF-anti-troponin I and F-anti-myoglobin, both at a concentration in 5ug/ml in PBS buffer with 10% sucrose, 1 mg/ml BSA and 1% sodium cholatedetergent is dispensed in spot 1. Spot 1 is designated the bindingreagent zone (diffusively bound). A 3 μl aliquot of Cy5-anti F at 5ug/ml in PBS with 10% sucrose, 1 mg/ml BSA, and 1% sodium cholate isdispensed in spot 4. Spot 4 is designated as the signal reagent zone.The back wall, still in the horizontal position, is then placed in a 37C convection oven and allowed to dry of 1 hour. Epoxy is then placedalong the rim to the back wall and the front wall is bounded to the backwall to assemble the unitized cuvette. The cuvette is stored desiccateduntil use.

To perform the sequential assay, 5 μl of a sample containing humanmyoglobin and troponin is dispensed at the bottom of the cuvette with amicropipette. 45 μl of assay buffer is added, sufficient to immerse bothcapture and binding reagent zones with the fluid remaining below thesignal reagent zone. To facilitate mixing and diffusion of the bindingreagent, 20 μl of the mixture is withdrawn and dispensed rapidly backinto the consumable with a micropipette, repeating several times over a10 second period. The assay mixture then incubates for 10 minutes atroom temperature, at which point the assay mixture is removed followedby 4 wash cycles with 70 μl of assay buffer at each cycle, keeping thefluid level below the signal reagent zone. After the last wash cycle,100 μl of assay buffer is added to the consumable, sufficient to immersethe signal reagent zone. To facilitate mixing and diffusion of thesignal reagent, 20 μl of the mixture is withdrawn and dispensed rapidlyback into the cuvette with a micropipette, repeating several times overa 10 second period. The assay mixture then incubates for 10 minutes atroom temperature, at which point the mixture is removed followed by 4wash cycles with 120 μl of assay buffer at each cycle. After the lastwash cycle, 120 μl of assay buffer is added to the cuvette and the Cy 5fluorescence is detected in spots 2 & 3 to respectively measure troponinI and myoglobin in the sample.

Example 5 Two-Step Sequential Protocol for Antibody Detection

With the back wall in the horizontal position, 3 μl of HBcAg at 1 ug/mlin PBS is dispensed in spot 2 and 3 μl of HBsAg at 1 ug/ml in PBS isdispensed in spot 3. Spots 2 & 3 are designated as the capture zone(non-diffusively bound). The back wall is then placed in a humidifiedchamber and the protein allowed to be adsorbed to the plastic forovernite at room temperature. The protein solution is then removed fromspots 2 & 3 with a micropipette. A wash cycle is then performed byadding 3 μl of assay buffer comprised of PBS, 1 mg/ml BSA and 0.1% Tween20 to each of the spots. After about 10 seconds the buffer is removed.The wash cycle is repeated 4 times. After the last wash cycle 3 μl of aPBS buffer containing 10% sucrose and 1 mg/ml BSA is placed in each ofthe spots. Following a 30 second incubation, the sucrose/PBS buffer isremoved from the spots leaving a thin fluid film on the surface of thespots. A 3 μl aliquot of Cy5-anti Ig(G & M) at 5 ug/ml in PBS with 10%sucrose, 1 mg/ml BSA, and 1% sodium cholate is dispensed in spot 4. Spot4 is designated as the signal reagent zone (diffusively bound). The backwall, still in the horizontal position, is then placed in a 37 Cconvection oven and allowed to dry of 1 hour. Epoxy is then placed alongthe rim to the back wall and the front wall is bounded to the back wallto assemble the unitized cuvette. The cuvette is stored desiccated untiluse.

To perform the sequential antibody assay, 5 μl of a sample containinganti HBcAg and anti HBsAg is dispensed at the bottom of the cuvette witha micropipette: 45 μl of assay buffer is added, sufficient to immersethe capture reagent zone with the fluid remaining below the signalreagent zone. To facilitate mixing, 20 μl of the mixture is withdrawnand dispensed rapidly back into the consumable with a micropipette,repeating several times over a 10 second period. The assay mixture thenincubates for 10 minutes at room temperature, at which point the assaymixture is removed followed by 4 wash cycles with 70 μl of assay bufferat each cycle, keeping the fluid level below the signal reagent zone.After the last wash cycle, 100 μl of assay buffer is added to theconsumable, sufficient to immerse the signal reagent zone. To facilitatemixing and diffusion of the signal reagent, 20 μl of the mixture iswithdrawn and dispensed rapidly back into the cuvette with amicropipette, repeating several times over a 10 second period. The assaymixture then incubates for 10 minutes at room temperature, at whichpoint the mixture is removed followed by 4 wash cycles with 120 μl ofassay buffer at each cycle. After the last wash cycle, 120 μl of assaybuffer is added to the cuvette and the Cy 5 fluorescence is detected inspots 2 & 3 to respectively measure anti HBcAg and anti HBsAg in thesample.

1. An immunoassay cuvette comprising a transparent front wall, a backwall, side walls, a bottom, and a top opening; the back wall has asubstantially planar surface made of a non-porous material comprising acapture zone and a signal reagent zone; the capture zone comprises oneor more spots each non-diffusely bound with a first member of a bindingpair; and the signal reagent zone has one or more spots each diffuselybound with a signal reagent comprising the first member or a secondmember of the binding pair.
 2. The immunoassay cuvette according toclaim 1, wherein the signal reagent comprises the first member of thebinding pair.
 3. The immunoassay cuvette according to claim 1, whereinthe signal reagent comprises the second member of the binding pair. 4.The immunoassay cuvette according to claim 1, wherein the first memberor the second member of the binding pair is labeled with a signalgenerating molecule.
 5. The immunoassay cuvette according to claim 1,wherein the signal generating molecule is a fluorescent dye, achemiluminescent dye, or an enzyme.
 6. The immunoassay cuvette accordingto claim 1, wherein the signal reagent zone is located above the capturezone.
 7. The immunoassay cuvette according to claim 1, wherein thebinding pair is an antigen and antibody against the antigen, a ligandand a receptor, or complementary strands of nucleic acids.
 8. Theimmunoassay cuvette according to claim 1, wherein the aspect ratio ofthe height to the width or the height to the thickness of the cuvette isat least 3 to
 1. 9. The immunoassay cuvette according to claim 1,wherein the non-porous material is polymethmethacrylate, polystyrene,polycarbonate, glass, quartz, or combination thereof.
 10. Theimmunoassay cuvette according to claim 1, wherein the back wall is blacktinted.
 11. An immunoassay cuvette comprising a transparent front wall,a back wall, side walls, a bottom, and a top opening; the back wall hasa substantially planar surface made of a non-porous material comprisinga capture zone, a binding reagent zone, and a signal reagent zone; thecapture zone comprises one or more spots each non-diffusely bound with afirst antibody against a defined antigen; the binding reagent zonecomprises one or more spots each diffusely bound with a binding reagentcomprising (a) the defined antigen or (b) a second antibody against thedefined antigen; the signal reagent zone has one or more spots eachdiffusely bound with a signal reagent comprising a conjugate of a signalgenerating molecule and a binding molecule that binds to the bindingreagent but does not bind to the first antibody; wherein the signalreagent zone is located above the binding reagent zone and the capturezone.
 12. The immunoassay cuvette according to claim 11, wherein thebinding reagent comprises the defined antigen.
 13. The immunoassaycuvette according to claim 11, wherein the binding reagent comprises thesecond antibody against the defined antigen.
 14. The immunoassay cuvetteaccording to claim 11, wherein (a) and (b) is labeled with a member of abinding pair.
 15. The immunoassay cuvette according to claim 11, whereinthe binding reagent zone is located above the capture zone.
 16. Theimmunoassay cuvette according to claim 11, wherein the aspect ratio ofthe height to the width or the height to the thickness of the cuvette isat least 3 to
 1. 17. The immunoassay cuvette according to claim 11,wherein the non-porous material is polymethmethacrylate, polystyrene,polycarbonate, glass, quartz, or combination thereof.